Five-Hour Detection of Intestinal Colonization with Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae Using the β-Lacta Phenotypic Test: the BLESSED Study

ABSTRACT Extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-PE) intestinal colonization is of particular concern as it negatively impacts morbidity and is the main source of external cross-contamination in hospitalized patients. Contact isolation strategies may be caught out due to the turnaround time needed by laboratories to report intestinal colonization, during which patients may be inappropriately isolated or not isolated. Here, we developed a protocol combining enrichment by a rapid selective subculture of rectal swab medium and realization of a β-Lacta test on the obtained bacterial pellet (named the BLESSED protocol). The performances of this protocol were validated in vitro on 12 ESBL-PE strains spiked into calibrated sample suspensions and confirmed in clinical settings using 155 rectal swabs, of which 23 (reference method) and 31 (postenrichment broth culture) came from ESBL-PE carriers. In vitro, the protocol detected, with 100% sensitivity, the presence of the 12 ESBL-PE strains from 104 CFU/mL. In the clinical validation cohort, 22 out of the 23 (reference method) and 28 out of the 31 (postenrichment broth culture) ESBL-PE-positive rectal samples were accurately detected. The diagnostic performances for ESBL-PE detection, considering all ESBL-PE carriers, were 90% sensitivity, 98% specificity, an 87% positive predictive value, and a 98% negative predictive value. Our protocol is a rapid and low-cost method that can detect intestinal colonization with ESBL-PE in less than 5 h more accurately than the reference method, opening the field for further studies assessing a rapid and targeted isolation strategy applied only to patients with a positive BLESSED protocol result. IMPORTANCE To both improve the efficiency of contact isolation among ESBL-PE carriers and avoid the unnecessary isolation of noncolonized patients, we should reduce the turnaround time of ESBL screening in laboratories and improve the sensitivity of diagnostic methods. The development of rapid and low-cost methods that satisfy these two goals is a promising approach. In this study, we developed such a technique and report its good diagnostic performance, opening the door for further studies assessing a rapid and targeted isolation strategy applied in a few hours only for patients truly colonized with ESBL-producing bacteria.

This is important since the study by Blanc et al used 48-72h protocols. It may be a little misleading to just point out the 100% sensititvity, when the comparisons in Table 4 and 5 show something different.
Reviewer #2 (Comments for the Author): In this manuscript, Gallah et al. describe the performance of a rapid diagnostic assay to detect ESBL-producing organisms in the gastrointestinal tract (GI) compared to culture on a chromogenic agar with and without enrichment broth. I hope the authors view these comments as being constructive and designed to help strengthen their manuscript.
Major Comments: Introduction: The first section of this manuscript includes a few lines of introducing the readers to the β-LACTA test (lines 62-65, but there is no information on how the test functions. Is it a molecular test, antigenic test, phenotypic, enzymatic, colorimetric? Please provide a brief description of the β-LACTA test. Methods: Part of the technical optimization procedure could be moved to the main body of the manuscript as it gives important insight as to how the test functions. The methods section should include a more detailed description of how the β-LACTA test works. Discussion: Recommend adding discussion for clinical labs on how they can implement this test.
Reviewer #3 (Comments for the Author): In the current study, the authors developed a rapid selective enrichment subculture method prior to the commercial β-LACTA{trade mark, serif} test (Bio-Rad Laboratories) to detect the intestinal colonization of Extended-Spectrum Beta-Lactamase-producing Enterobacteriaceae (ESBL-PE) from rectal swab. However, the authors did not carry out a head-to-head experiment to compare the performance of β-LACTA{trade mark, serif} test alone and enrichment subculture with β-LACTA{trade mark, serif} test. Although detection of ESBL-PE is a clinically important issue, it is hard to say that this research has new breakthroughs. In my opinion, I think it's just a small modification for the original protocol. Major: 1. Native English editing recommended 2. The term" digestive colonization" could be changed to intestinal colonization or intestinal colonization of ESBL-PE. Minor: 1. Add the full name of "BLT test" "ESBL-PE" in the supplementary methods.

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I have reviewed the paper by Gallah et al.
Although English is correct, some changes in the writing style may help the manuscript to be more readable.
The introduction needs to briefly describe what the beta-LACTA test is.
Methods for the blessed protocol need to state the number of patients used.
Ideally the reader would like to see a growth curve with the sensitivity of the b-LACTA detection. This hasn't been shown. Otherwise, how do we know the 4 hour shaking in BHI was optimal? I understand the data is somehow "in there" from what is shown in Table 1.
This is important since the study by Blanc et al used 48-72h protocols.
It may be a little misleading to just point out the 100% sensititvity, when the comparisons in Table 4 and 5 show something different.
We thank the editor and the reviewers for their comments and the opportunity to revise and improve our manuscript. Please find below our point-by-point responses to these comments.

Reviewer #1
1. I have reviewed the paper by Gallah et al. Although English is correct, some changes in the writing style may help the manuscript to be more readable.
We thank the reviewer for this suggestion. The revised version of the manuscript has been entirely proofread and corrected by a native English speaker.

The introduction needs to briefly describe what the beta-LACTA test is.
According to this comment and to comment #2 of reviewer #2, we added two sentences briefly explaining how the β-LACTA test functions, as follows: "In contrast, phenotypic tests are simple to use and less expensive. The β-LACTA™ test (BLT) (Bio-Rad Laboratories, Marnes-La-Coquette, France) is a phenotypic test based on the cleavage of a chromogenic cephalosporin, turning the substrate from yellow to red in the presence of GNB strains producing a beta-lactamase able to hydrolyze 3rd generation cephalosporins. Thus, the test turns positive mainly in the presence of ESBL-PE; however, the test also reacts to some carbapenemase-producing and AmpC-hyperproducing GNB. BLT has shown high diagnostic performance for the rapid identification of ESBL-PE when performed on isolated bacterial colonies (17,18) or directly on bacterial pellets from urine or bronchial aspirate samples (12,14). However, it has never been assessed for the rapid detection of ESBL-PE intestinal colonization." (lines 69-77 of the revised manuscript).
3. Methods for the blessed protocol need to state the number of patients used.
The number of patients (and rectal swabs) on which the BLESSED protocol was validated in clinical settings is reported in the "clinical validation" section of the results, first sentence of the "Population" paragraph: "A total of 155 rectal swabs from 155 different patients in the ICU were collected." (line 165 of the original manuscript, line 184 of the revised version). According to the reviewer's comment, we have also added this information in the method section (section "evaluation of the BLESSED protocol in clinical settings, "collection" paragraph), as follows: "Unused residual media for laboratory diagnosis from rectal eSwab™ performed to screen the intestinal colonization with multidrug-resistant GNB of 155 patients hospitalized in the ICUs of two university hospitals […]" (lines 122-124 of the revised manuscript).
4. Ideally the reader would like to see a growth curve with the sensitivity of the b-LACTA detection. This hasn't been shown. Otherwise, how do we know the 4-hour shaking in BHI was optimal? I understand the data is somehow "in there" from what is shown in Table 1. This is important since the study by Blanc et al used 48-72h protocols.
We thank the reviewer for this suggestion. We agree that our protocol presents the advantage of providing results faster than Blanc et al. protocol. Indeed, Blanc's protocol was performed on fresh colonies obtained after overnight culture on selective agar plates, leading to turnaround time of 36-38h to report ESBL-PE positive sample when our protocol allows identification of ESBL-PE from rectal swabs in 5 hours.
The optimal four-hour incubation time of our BLESSED protocol was determined in preliminary experiments. The reviewer is right, we did not show these preliminary results in the original manuscript. We only mentioned that direct realization of a β-LACTA™ test (BLT) on the pellet obtained from centrifugation of the rectal swab medium (i.e., without any incubation in the selective enrichment broth) led to insufficient performances. However, it is not so simple to represent in one figure the growth curve of BLT sensitivity as requested but we added figures below that explained our best choice. Indeed, we assessed many experimental conditions including various bacterial inoculums, incubation times of the selective broth, and incubation times in the BLT reagents before reading the results. We have progressed by steps to arrive at the final BLESSED protocol. In particular, when a condition showed poor performance after the first set of experiments, it was dropped to refine the protocol to its final version. Consequently, the number of technical development experiments was not strictly the same for all experimental conditions. However, we changed several parts of the manuscript and of the supplementary methods file, to better explain why we have retained the four-hour incubation time of the selective broth and the 60- 5. It may be a little misleading to just point out the 100% sensitivity, when the comparisons in Table 4 and 5 show something different.
We have carefully checked the content of Tables 4 and 5, and we did not see incoherent results. Indeed, Table 3 contains the results of the clinical evaluation of the BLESSED protocol when compared with the culture of the rectal swab medium on ESBL chromID® agar plate (the reference method). These results are summarized in the second column of Table 5 ("Compared with rectal swab culture"). Along these lines, sensitivity was calculated from 22 samples positive with the BLESSED protocol among the 23 samples showing an ESBL-PE in culture of the rectal swab medium = 22/23 = 96%, as indicated in Table 5. Then, Table 4 contains the results of the clinical evaluation of the BLESSED protocol when compared with the culture of the post-enrichment broth on ESBL chromID® agar plate. Indeed, we observed that more ESBL-PE containing samples were detected when culturing the post-enrichment broth, as a consequence of a lack of sensitivity of the reference method. Thus, this time, sensitivity was calculated from 28 samples positive with the BLESSED protocol among the 31 samples showing an ESBL-PE after culture of the post-enrichment broth = 28/31 = 90%, as indicated in the third column of Table 5. And so on, with calculations of specificity, and positive and negative predictive values. Finally, we did not report a "100% sensitivity", except in the sentence of the result section reporting the results of the in vitro performance of the BLESSED protocol: "however, it was positive for all 12 ESBL-PE strains spiked at the 10 4 CFU/mL inoculum (100% sensitivity)" (lines 159-160 of the original version and 178-179 of the revised version). Further, in the first paragraph of the discussion, we already stated: "Thus, considering in vitro and clinical evaluations, the BLESSED protocol had a sensitivity of more than 95% in detecting an ESBL-PE inoculum ≥10 4 UFC/ml" (lines 213-215 of the original version and 233-235 of the revised version), not pointing out "100% sensitivity". Consequently, we believe we have not overstated the interpretation of our results.

Reviewer #2
1. In this manuscript, Gallah et al. describe the performance of a rapid diagnostic assay to detect ESBLproducing organisms in the gastrointestinal tract (GI) compared to culture on a chromogenic agar with and without enrichment broth. I hope the authors view these comments as being constructive and designed to help strengthen their manuscript.
We thank the reviewer for this comment and we assure her/him that we have taken her/his remarks as constructive elements.
Major Comments: 2. Introduction: The first section of this manuscript includes a few lines of introducing the readers to the β-LACTA test (lines 62-65), but there is no information on how the test functions. Is it a molecular test, antigenic test, phenotypic, enzymatic, colorimetric? Please provide a brief description of the β-LACTA test.
Following this comment and comment #2 of reviewer #1, we added two sentences briefly explaining how the β-LACTA test functions, as follows: "In contrast, phenotypic tests are simple to use and less expensive. The β-LACTA™ test (BLT) (Bio-Rad Laboratories, Marnes-La-Coquette, France) is a phenotypic test based on the cleavage of a chromogenic cephalosporin, turning the substrate from yellow to red in the presence of GNB strains producing a beta-lactamase able to hydrolyze 3rd generation cephalosporins. Thus, the test turns positive mainly in the presence of ESBL-PE; however, the test also reacts to some carbapenemase-producing and AmpC-hyperproducing GNB. BLT has shown high diagnostic performance for the rapid identification of ESBL-PE when performed on isolated bacterial colonies (17,18) or directly on bacterial pellets from urine or bronchial aspirate samples (12,14). However, it has never been assessed for the rapid detection of ESBL-PE intestinal colonization." (lines 69-77 of the revised manuscript).
3. Methods: Part of the technical optimization procedure could be moved to the main body of the manuscript as it gives important insight as to how the test functions. The methods section should include a more detailed description of how the β-LACTA test works.
Following the reviewer's comment, we fully reworded the first paragraph of the "technical optimization procedure" section. We added some sentences from the Supplementary Methods file, and better explained how the BLT works, as follows: "We first assessed the performance of BLT directly performed on bacterial pellets obtained from the centrifugation of 1 mL of rectal swab medium (see Supplementary Methods online for additional details). The pellets were completely homogenized in one drop (approximately 50 μL) of each reagent of the BLT assay and incubated at 37 °C. This "direct" procedure showed poor diagnostic performance in detecting ESBL-PE (sensitivity 60%, 95% confidence interval (CI) (26%-88%), specificity 58% (42%-72%), positive predictive value 24% (15%-37%), and negative predictive 87% (75%-94%)). These results can be explained by the low sensitivity in detecting ESBL activity diluted in the swab transport medium and by the low specificity due to the false positive result related to other bacteria that can turn the BLT positive, such as some bacteria of the anaerobic flora. We then developed a rapid selective subculture technique to increase both the sensitivity and specificity of the procedure." (lines 83-93 of the revised version). We hope that the reviewer will understand that we had to present results (about the diagnostic performance) of this very preliminary phase (which is no longer mentioned in the rest of the manuscript) in a section dedicated to methods.
4. Discussion: Recommend adding discussion for clinical labs on how they can implement this test.
We thank the reviewer for this suggestion. According to this comment we added a few sentences at the end of the discussion with regards to the possible implementation of this protocol in the daily practice of clinical labs, as follows: "Second, our protocol requires several manipulations. Although their number is quite limited, it could be hypothesized that the automation of the procedure would increase its diffusion. However, this must be balanced with the fact that the reference screening strategy is also not fully automated. Indeed, plating of rectal swab medium on selective agar plates, reading the culture results of these plates, and, in case of positivity, sub-culturing of isolated strains to determine their antibiotic susceptibility remain mostly manual. Thus, the BLESSED protocol could probably already be implemented in most laboratories, without a significant increase in technical handling time, but allowing the detection of ESBL-PE intestinal colonization from patient sampling to final results in less than a working day for a laboratory technician." (lines 290-299 of the revised version).